Electrode and Use Thereof

ABSTRACT

The application relates to an electrode for use in the electrochemical detection of a target species, wherein the electrode has a planar surface disposed on which are probe molecules that are capable of binding selectively to the target species, wherein the electrode, prior to binding of the probe molecules with the target species, has an electron transfer resistance per area of the electrode of from 10 megaohms cm −2  to 95 megaohms cm −2 .

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/398,208, filed Oct. 31, 2014, which is a National Stage Applicationunder 35 U.S.C. 371 of co-pending PCT application numberPCT/GB2013/051121, filed 1 May 2013; which claims priority toGB1207585.9, filed 1 May 2012, each of which are hereby incorporated byreference in their entireties for any and all non-limiting purposes.

FIELD OF THE INVENTION

The present invention relates to an electrode for use in theelectrochemical detection of target species, including, but not limitedto, C-reactive protein.

BACKGROUND

C-reactive protein (CRP) is an acute-phase protein synthesized by theliver, widely accepted as a biomarker for cardiovascular disease andinflammation (May and Wang 2007, Miller et al. 2007, Mygind et al. 2011,Pai et al. 2008). Generally, levels in plasma are less than 2.0 mg/L forhealthy individuals (Vikholm-Lundin and Albers 2006), but increase up to1000 fold during an acute phase of inflammation (Gabay and Kushner1999). The American Heart Association and the United States Centre forDisease Control have suggested three categories of CRP concentration forthe evaluation of cardiovascular disease risk: a CRP concentration below1.0 mg/L representing low risk, a 1.0 to 3.0 mg/L range average risk,and levels above 3.0 mg/L representing high risk (Kushner and Sehgal2002, Lee et al. 2011). The reliable and early quantification of thistarget if often, then, cited as a means of improving the outcome ofcardiovascular or inflammatory disease through appropriate interventionor treatment.

Currently, a number of CRP testing methods are available in clinicallaboratories using turbidimetric and nephelometric technologies (Robertset al. 2000, Roberts et al. 2001), or human CRP enzyme-linkedimmunosorbent assay (ELISA) kits. However, these methods are generallynot suitable for the clinical practice as they are either not sensitiveenough, time-consuming, prone to false negatives or cost-ineffective(Pearson et al. 2004). CRP quantification methods based on surfaceplasmon resonance (SPR) (Hu et al. 2006, Meyer et al. 2006),piezoelectric microcantilevers (Wee et al. 2005), quartz crystalmicrobalance technology (Kim et al. 2009), microfluidics (Lee et al.2011) and electrochemistry (Buch and Rishpon 2008, Centi et al. 2009),have been developed during the past few years. Among these,electrochemical assays promise most in terms of low cost, flexibilityand sensitivity. Electrochemical impedance spectroscopy (EIS), inparticular, can sensitively monitor the changes in capacitance orcharge-transfer resistance associated with material binding atspecifically prepared receptive electrode surfaces and requires no priorlabelling (Bogomolova et al. 2009, Rodriguez et al. 2005). In recentyears a number of CRP assays by EIS have been reported. To date,however, these have been either of limited sensitivity (Vermeeren et al.2011), not demonstrably specific (Hennessey et al. 2009), or to notencompass a clinically relevant range (Chen et al. 2008, Qureshi et al.2010).

This subject-matter of this application relates to the development of arobust and highly sensitive assay for CRP in whole and dilute bloodserum across the entire clinically relevant range. The technique canalso be applied to other markers. The interfaces are readily prepared,exhibit very good selectivity and are re-useable after assay with noapparent loss of sensitivity. We have, additionally, considered theimportance of receptive layer initial resistance in subsequentlyobserved sensitivity and demonstrated not only a clear correlation butalso an ability to tune, and therefore optimise, receptive filmcharacteristics.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Nyquist plots of different electrodes recorded in PBST (10mM, pH 7.4) solution containing 1.0 mM Fe(CN)₆ ^(3−/4−), for (a) baregold electrode; (b) gold electrode modified with the self-assembledmonolayer; and (c) gold electrode with CRP antibody immobilized on themonolayer. The inset magnifies the high frequency region.

FIG. 2A is a graph showing the effect of CRP antibody immobilizationtime on the initial charge transfer resistance, R_(ct) and sensitivityof the biosensor (the sensitivity was determined as change in R_(ct)divided by the initial R_(ct)); and FIG. 2B is a graph illustrating therelationship between the initial R_(CT) and the sensitivity.

FIG. 3A is a graph showing the recorded charge-transfer resistance(R_(ct)) of the biosensor after the incubation with differentconcentrations of CRP protein in PBST (10 mM, pH 7.4) containing 1.0 mMFe(CN)₆ ^(3−/4−); and FIG. 3B is a graph showing Faradaic impedancespectra corresponding to the biosensor after the incubation in PBSTsolution with different CRP concentrations (curves i to viii represent0, 0.5, 1.0, 2.0, 5.0, 10, 25, and 50 nM CRP, respectively).

FIG. 4A is a graph showing calibration curves for the CRP biosensorsperformed in different solutions, namely:

-   -   (i) PBST (10 mM, pH 7.4) with 1.0 mM Fe(CN)₆ ^(3−/4−);    -   (ii) PBST (10 mM, pH 7.4) with 1.0 mM Fe(CN)₆ ^(3−/4−) and 10%        human blood serum; and    -   (iii) PBST (10 mM, pH 7.4) with 1.0 mM Fe(CN)₆ ^(3−/4−) and 20%        human blood serum;        And FIG. 4B is a graph showing Faradaic impedance spectra        corresponding to the biosensor after the incubation in PBST        solution containing 1.0 mM Fe(CN)₆ ^(3−/4−) and 10% human blood        serum, with different CRP concentrations (curves i to viii        represent 0, 0.5, 1.0, 2.0, 5.0, 10, 25, and 50 nM CRP,        respectively).

FIG. 5 shows a comparison of biosensor assays of CRP in buffer solutionand pure blood serum. Biosensors were incubated in CRP spiked buffersolution and pure blood serum, respectively, and impedance analysescarried out in PBST solution containing 1.0 mM Fe(CN)₆ ^(3−/4−).Impedance levels of CRP free buffer or pure blood serum were taken asbackground.

FIG. 6 shows the regeneration of the CRP biosensor. The biosensor wasregenerated by immersing the electrode in 6 mM NaOH and 0.6% ethanol for5 min and then washed with PBST, and the impedance measurements weretaken in a solution containing PBST (10 mM, pH 7.4), 1.0 mM Fe(CN)₆^(3−/4−) and 10% human blood serum.

FIG. 7 is a schematic illustration of the stepwise fabrication of CRPresponsive EIS interfaces.

FIG. 8 shows an equivalent circuit model that can be used for datafitting to determine the charge transfer resistance of an electrode asdescribed herein.

SUMMARY OF THE INVENTION

In a first aspect, there is provided an electrode for use in theelectrochemical detection of a target species, wherein the electrode hasa planar surface disposed on which are probe molecules that are capableof binding selectively to the target species, wherein the electrode,prior to binding of the probe molecules with the target species, has anelectron transfer resistance per area of the electrode of from 10megaohms cm⁻² to 95 megaohms cm⁻². In an embodiment, the target speciesis or comprises C-reactive protein and/or the probe molecules compriseantibodies or antibody fragments. The use with other target species andprobe molecules is nevertheless described below.

In an embodiment, there is provided an electrode for use in theelectrochemical detection of C-reactive protein, wherein the electrodehas a planar surface disposed on which are probe molecules that arecapable of binding selectively to C-reactive protein, wherein theelectrode prior to binding of the probe molecules with C-reactiveprotein has an electron transfer resistance per area of the electrode offrom 10 megaohms cm⁻² to 95 megaohms cm⁻²

In an embodiment, there is provided an electrode for use in theelectrochemical detection of a target species, wherein the electrode hasa planar surface disposed on which are probe molecules comprisingantibodies or antibody fragments that are capable of binding selectivelyto the target species, wherein the electrode prior to binding of theprobe molecules with the target species has an electron transferresistance per area of the electrode of from 10 megaohms cm⁻² to 95megaohms cm⁻²

In a third aspect, there is provided a method for detecting a targetspecies in an electrochemical impedance spectroscopy technique, whereinthe method comprises contacting an electrode defined in the first aspectwith a carrier medium comprising the target species, and detecting anelectrical signal at the working electrode.

In a third aspect, there is provided an electrochemical impedancespectrometer, wherein the spectrometer comprises an electrode as definedin the first aspect.

In a fourth aspect, there is provided a use of an electrode according tothe first aspect or an electrochemical impedance spectrometer accordingto the third aspect for the detection of a target species.

The present inventors have found that controlling the resistance of anelectrode, which is related to the amount of coverage of a probemolecule, before binding to a target can significantly increase thesensitivity of the electrode. It has previously been assumed thatincreasing the coverage of probe molecules on a surface would increaseits sensitivity. However, the present inventors found that keeping thecoverage below certain levels (as indicated by initial resistance),increased the sensitivity of the electrode.

DETAILED DESCRIPTION

The present invention provides the first to the fourth aspects describedherein. Optional and preferred features will now be described. Any ofthe features described herein may be combined with any of the otherfeatures described herein, unless otherwise stated.

The electrode, prior to binding of the probe molecules with the targetspecies, preferably has an electron transfer resistance per area of theelectrode of 95 megaohms cm⁻² or less. The electrode, prior to bindingof the probe molecules with the target species, preferably has anelectron transfer resistance per area of the electrode of 10 megaohmscm⁻² or more. The electrode, prior to binding of the probe moleculeswith the target species, preferably has an electron transfer resistanceper area of the electrode of from 10 megaohms cm⁻² to 95 megaohms cm⁻².

Optionally, the electrode, prior to binding of the probe molecules withthe target species, has an electron transfer resistance per area of theelectrode of from 20 megaohms cm⁻² to 95 megaohms cm⁻², optionally anelectron transfer resistance per area of the electrode of from 40megaohms cm⁻² to 95 megaohms cm⁻², optionally an electron transferresistance per area of the electrode of from 50 megaohms cm⁻² to 95megaohms cm⁻², optionally an electron transfer resistance per area ofthe electrode of from 55 megaohms cm⁻² to 95 megaohms cm⁻².

Electron transfer resistance, sometimes termed the charge transferresistance, can be measured using known techniques. In an embodiment,the electron transfer resistance is determined by using anelectrochemical impedance spectrometer to obtain impedance informationabout the electrode, and using an ideal Randles equivalent circuit,which includes the charge transfer resistance as an element of thecircuit. Such a Randles equivalent circuit is described, for example, inVyas R N, Li K Y, Wang B. 2010. Modifying Randles Circuit for Analysisof Polyoxometalate Layer-by-Layer Films. Journal of Physical Chemistry B114: 15818-15824, which is incorporated herein by reference. An exampleof an ideal Randles equivalent circuit is illustrated in FIG. 8. In thisfigure, R_(ct) represents the charge (or electron) transfer resistance,Z_(w) represents the Warburg impedance, R_(s) is the solution resistance(sometimes denoted R_(sol)), and Cat is the capacitance between theelectrode surface and ions in the liquid carrier medium (this issometimes denoted C_(surf)). Fitting impedance data to a circuit toobtain electron (or charge) transfer resistance is known to the skilledperson. It is described in many publications, including, but not limitedto, Electroanalysis 19, 2007, No. 12, 1239-1257 (an article entitledLabel-free Impedance Biosensors: Opportunities and Challenges, authoredby Daniels, et al), which is incorporated herein by reference in itsentirety, and references cited therein. Commercial software is availablefor circuit fitting, for example “Fit and Simulation version 1.7”software available from Autolab, The Netherlands, which may accompany afrequency resolved analyser module of an EIS spectrometer (e.g. alsoavailable from Autolab, The Netherlands).

The electron transfer resistance per area of the electrode is determinedby dividing the charge transfer resistance of the electrode by the areaof the electrode (having the probe molecules thereon). This area may bedetermined using known techniques. In an embodiment, the area of theelectrode is the effective surface area of the electrode, which can becalculated by taking the area of a cathodic peak in coulombs andapplying the relationship

${A = \frac{Q}{482\mspace{14mu} {\mu C}\mspace{14mu} {cm}^{- 2}}},$

where A is the effective area of the electrode, and Q is the area of thecathodic peak in colombs (see, for example, Hoogvliet, J. C.; Dijksma,M.; Kamp, B.; van Bennekom, W. P. Anal Chem 2000, 72, 2016, which isincorporated herein by reference in its entirety), and dividing thecharge transfer resistance (Ret) by the effective area.

The electrode may comprise any electrically conducting material. Theworking electrode may comprising a metal or carbon. The metal may be ametal in elemental form or an alloy of a metal. Optionally, the whole ofthe electrode comprises a metal or carbon. The electrode may comprise atransition metal. The electrode may comprise a transition metal selectedfrom any of groups 9 to 11 of the Periodic Table. The electrode maycomprise a metal selected from, but not limited to, rhenium, iridium,palladium, platinum, copper, indium, rubidium, silver and gold. Theelectrode may comprise a metal selected from gold, silver and platinum.The electrode may comprise a carbon-containing material, which may beselected from edge plane pyrolytic graphite, basal plane pyrolyticgraphite, glassy carbon, boron doped diamond, highly ordered pyrolyticgraphite, carbon powder and carbon nanotubes.

In a preferred embodiment, the electrode comprises gold.

The surface of the electrode is planar, which includes a generally flatsurface, typically without indentations, protrusions and pores. Suchelectrode surfaces can be prepared, before probe molecules and anyassociated linker molecules are bound to the surface, by techniques suchas polishing with fine particles, e.g. spraying with fine particles,optionally in a sequence of steps where the size of the fine particlesis decreased in each polishing step. The fine particles may, forexample, comprise a carbon-based material, such as diamond, and/or mayhave particles with diameters of 10 μm or less, optionally 5 μm or less,optionally 3 μm or less, optionally 1 μm or less, optionally 0.5 μm orless, optionally 0.1 μm or less. Following polishing, the electrodesurface may be washed, e.g. ultrasonically, optionally in a suitableliquid medium, such as water, e.g. for a period of at least 1 minute,e.g. from about 1 minute to 10 minutes. Optionally, the electrodesurface may be washed with an abrasive, e.g. acidic, solution, forexample following the polishing and, if used, ultrasonic washing steps.The abrasive solution may comprise an inorganic acid, e.g. H₂SO₄, and/ora peroxide, e.g. H₂O₂, in a suitable liquid medium, e.g. water.Optionally, the electrodes can be electrochemically polished, which mayfollow any steps involving one or more of polishing with fine particles,washing e.g. ultrasonically and/or using an abrasive solution. Theelectrochemical polishing may involve cycling between an upper and lowerpotential until a stable reduction peak is reached, e.g. an upperpotential of 0.5 V or more, optionally 1 V or more, optionally 1.25 V ormore, and a lower potential of 0.5 V or less, optionally 0.25 V or less,optionally 0.1 V or less.

The probe molecules are capable of binding selectively to a targetspecies, which may be or comprise C-reactive protein. Other targetspecies are described below. The probe molecule preferably comprises abinding species selected from an antibody, an antibody fragment, anaptamer, an oligosaccharide, a peptide, and a protein. Preferably, theprobe molecules comprise a binding species selected from one or more ofan antibody, a nucleic acid and a peptide. The binding species iscapable of binding to the target species, e.g. C-reactive protein. Theprobe moieties bind selectively to the target species, e.g. C-reactiveprotein.

If the probe molecules comprise an antibody or an antibody fragment, theantibody or the antibody fragment may be selected from one or more ofthe classes IgA, IgD, IgE, IgG and IgM. In a preferred embodiment, theantibody or antibody fragment is of the IgG type. The antibody bindsselectively to the target species. The antibody or antibody fragment maybe derived from a mammal, including, but not limited to, a mammalselected from a human, a mouse, a rat, a rabbit, a goat, a sheep, and ahorse. In an embodiment, the probe molecules comprise an antibody of theIgG type derived from a goat.

If the probe molecules comprise an aptamer, the aptamer may be selectedfrom a peptide aptamer, a DNA aptamer and a RNA aptamer.

The target species may be selected from, but is not limited to,proteins, polypeptides, antibodies, nanoparticles, drugs, toxins,harmful gases, hazardous chemicals, explosives, viral particles, cells,multi-cellular organisms, cytokines and chemokines, ganietocyte,organelles, lipids, nucleic acid sequences, oligosaccharides, chemicalintermediates of metabolic pathways and macromolecules. In preferredembodiments, the target species comprises, consists essentially of, orconsists of, a biological molecule, more suitably a biologicalmacromolecule, most suitably a polypeptide.

If the target species is or comprise a protein, the protein may beselected from, but is not limited to, native proteins, denaturedproteins, protein fragments, and prokaryotically or eukaryoticallyexpressed proteins. Protein may have its normal meaning in the art, andmost preferably ‘protein’ refers to a polypeptide molecule. Suchpolypeptide may comprise modifications such as glycosylation;phosphorylation or other such modifications.

If the target species is an antibody, the antibody may be selected fromone or more of the classes IgA, IgD, IgE, IgG and IgM.

If the target species is a nanoparticle, the nanoparticle can beselected from, but is not limited to, one or more of insulating,metallic or semiconducting nanoparticles.

If the target species is a drug, the drug may be selected from, but isnot limited to, alcohol (e.g. ethanol), amphetamines, amyl nitrate,heroin, ketamine, anabolic steroids, LSD, solvents, cannabis, cocaine(such as cocaine hydrochloride or ‘coke’), tobacco, tranquilisers, crack(i.e. cocaine free base), ecstasy and/or gammhydroxybutyrate (GHB).Alternatively, in some embodiments, the drug may be a medicinalsubstance.

The target species may be a candidate drug, e.g. a chemical orbiological entity which may be tested or screened for a particularactivity or property using the present invention.

If the target species is a toxin, the toxin may be selected from, but isnot limited to, one or more toxins originating from animals, plants, orbacteria.

If the target species is a viral particle, the viral particle may beselected from, but is not limited to, one or more viral particles withand without a genome.

If the target species is a cell, the cell may be selected from, but isnot limited to, one or more of pluripotent progenitor cells, human cells(e.g. B-cells, T-cells, mast cells, phagocytes, neutrophils,eosinophils, macrophages, endothelial cells), cancerous cells (e.g.those originating from liver, cervical bone, pancreatic, colorectal,prostate, epidermal, brain, breast, lung, testicular, renal, bladdercancers), unicellular organisms of non-human origin, algae, fungi,bacteria, plant cells, parasite eggs, plasmodia and mycoplasma.

If the target species is an organelle, the organelle may be selectedfrom, but is not limited to, one or more of nucleus, mitochondria, Golgiapparatus, endoplasmic reticulum, lysosome, phagosome, intracellularmembranes, extracellular membranes, cytoskeleton, nuclear membrane,chromatin, nuclear matrix and chloroplasts.

If the target species is a lipid, the lipid may be selected from, but isnot limited to, one or more of signalling lipids, structural lipids,phospholipids, glycolipids and fatty acids.

If the target species is nucleic acid sequence, the nucleic acidsequence may be selected from, but is not limited to, one or more ofDNA, cDNA, RNA, rRNA, mRNA, miRNA and tRNA.

If the target species is an oligosaccharide, the oligosaccharide may beselected from, but is not limited to, one or more of oligosaccharides ofhuman, animal, plant, fungal or bacterial origin.

In a preferred embodiment, the target species is a protein. The methodand other aspects of the invention may be used for the detection oridentification of a proteins.

The target species may be any antigen or analyte that is indicative of aparticular disease. The target may be selected from, for example,C-reactive protein, angiotensin I converting enzyme(peptidyl-dipeptidase A) 1; adiponectin; advanced glycosylation endproduct-specific receptor; alpha-2-HS-glycoprotein; angiogenin,ribonuclease, RNase A family, 5; apolipoprotein A-1; apolipoprotein B(including Ag(x) antigen); apolipoprotein E; BCL2-associated X protein;B-cell CLL/lymphoma 2; complement C3; chemokine (C-C motif) ligand 2; CD14, soluble; CD 40, soluble; cdk5; pentraxin-related; cathepsin B;dipeptidyl peptidase IV; Epidermal growth factor; endoglin; Fas;fibrinogen; ferritin; growth hormone 1; alanine aminotransferase;hepatocyte growth factor; haptoglobin; heat shock 70 kDa protein 1 B;intercellular adhesion molecule 1; insulin-like growth factor 1(somatomedin C); insulin-like growth factor 1 receptor; insulin-likegrowth factor binding protein 1; insulin-like growth factor bindingprotein 2; insulin-like growth factor-binding protein 3; interleukin 18;interleukin 2 receptor, alpha; interleukin 2 receptor, beta; interleukin6 (interferon, beta 2); interleukin 6 receptor; interleukin 6 signaltransducer (gp130, oncostatin M receptor); interleukin 8; activin A;leptin (obesity homolog, mouse); plasminogen activator, tissue;proopiomelanocortin(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulatinghormone/beta-melanocyte stimulating hormone/beta-endorphin); proinsulin;resistin; selectin e (endothelial adhesion molecule 1); selectin P(granule membrane protein 140 kDa, antigen CD62); serpin peptidaseinhibitor, clade E (nexin, plasminogen activator inhibitor type 1),member 1; serum/glucocorticoid regulated kinase; sex hormone-bindingglobulin; transforming growth factor, beta 1 (Camurati-Engelmanndisease); TIMP metallopeptidase inhibitor 2; tumor necrosis factorreceptor superfamily, member 1 B; vascular cell adhesion molecule 1(VCAM-1); vascular endothelial growth factor; Factor II, Factor V,Factor VIII, Factor IX, Factor XI, Factor XII, F/fibrin degradationproducts, thrombin-antithrombin III complex, fibrinogen, plasminogen,prothrombin, and von Willebrand factor and the like. Markers useful fordiabetes include for example C-reactive protein; glucose; insulin; TRIG;GPT; HSPA1 B; IGFBP2; LEP; ADIPOQ; CCL2; ENG; HP; IL2RA; SCp; SHBG; andTIMP2.

The target species may be a target associated with monitoring diabetes.In an embodiment, the target may be selected from glucose, insulin,Interleukin 2 receptor alpha (IL2-RA), C-reactive protein (CRP) andglycated hemoglobin (HbAlc). If the target is glucose, the probemoieties may be selected from, for example, the molecular recognitionelement of GDH-FAD assay or a glucose/galactose binding protein (“GGBP”)(Scholle, et al., Mol. Gen. Genet 208:247-253 (1987)). If the target isIL-2RA, the probe moieties may comprise or consist of a monoclonalantibody specific for IL-2RA. If the target species is or comprisesC-reactive protein, preferably this is human C-reactive protein.

As indicated above, preferably, the probe molecules comprise a bindingspecies selected from one or more of an antibody, a nucleic acid and apeptide. The binding species may be directly attached to the surface ofthe electrode or attached to the surface of the electrode via a linkerspecies. If a linker species is present on the surface of the electrode,preferably the linker species forms a self-assembling monolayer.

The electrode as described herein may be formed by forming aself-assembling monolayer of linker species, optionally activating thelinker species, and then binding the binding species to at least some ofthe linker species.

Preferably, the electrode surface having the probe molecules thereon, asa whole, is selective for the target species. If the electrode surfacehaving the probe molecules thereon is selective for the target species,this indicates that substantially only or only the target species willbind to the surface (binding to the probe molecules), and other species(e.g. present in the carrier medium with the target species) will notbind, or not bind to any significant degree, to other parts of theelectrode surface or other species thereon. For example, the electrodesurface may comprise a self-assembling monolayer of linker molecules,some of which are bound to probe moieties, e.g. antibodies, thatselectively bind to a target (e.g. C-reactive protein). When in a liquidcarrier medium, e.g. blood, the electrode surface preferably only bindsto the target species, not to other species present in the liquidcarrier medium. Such selective electrode surfaces may be termed highlyselective electrode surfaces.

In an embodiment, the probe molecule is of the formula A--L--B, where Ais a moiety that binds to the surface of the electrode, L is a linkermoiety and B is a moiety which binds to the target species, e.g.C-reactive protein.

‘A’ may be selected from an appropriate binding group, depending on thenature of the material of the electrode. A may be selected from, but isnot limited to, biotin, hydrazine, alkynyl, alkylazide, amino, hydroxyl,carboxy, thio, aldehyde, phosphoinothioester, maleimidyl, succinyl,succinimidyl, isocyanate, ester, strepavidin, avidin, neuavidin, andbiotin binding proteins. If the electrode comprises a noble material,e.g. gold, silver or platinum, A is preferably thio, which may beselected from —SH and —S—. If the electrode comprises a metal that has alayer of oxide on its surface, e.g. copper, A may be a carboxy group.

L may be any species that covalently links A with B. L is preferably aspecies that allows formation of a self-assembling monolayer. L maycomprise an alkylene moiety comprising at least 2 carbons, the alkylenemoiety being directly attached to A; optionally the alkylene moiety is astraight-chain alkylene moiety. L may comprise an alkylene moietycomprising at least 10 carbons, optionally from 10 to 30 carbons,optionally from 10 to 20 carbons, optionally from 11 to 15 carbon atoms,and the alkylene moiety is optionally a straight-chain alkylene moiety,and the alkylene moiety is directly attached to A.

In an embodiment, L is of the formula —(CH₂)_(n)-(—O—CH₂—CH₂—)_(m)-D-,wherein n is from 1 to 30 and m is from 0 to 10 and D is a group thatbinds to B. D may be selected from a single bond, —(C═O)—, —OCH₂—(C═O)—,—(C═O)—NH—, —(C═O)—O—OCH₂—(C═O)—NH—, —OCH₂—(C═O)—OH—, —O—, —NH—. n maybe from 10 to 20. m may be 1 to 5, optionally 2 to 4, optionally 3.Optionally, if D is any one of the species (C═O)—NH—,—(C═O)—O—OCH₂—(C═O)—NH—, —OCH₂—(C═O)—O—, —O— and —NH—, then —NH— or —O—in these species may be derived from a probe molecules, e.g. antibody,prior to being bound to the linker species L.

B may be selected from a binding species as described above, for exampleselected from an antibody, an antibody fragment, an aptamer, anoligosaccharide, a peptide, a protein. Such species that bindselectively to target species, e.g. C-reactive protein, are availablecommercially, e.g. goat anti-human CRP polyclonal antibody, which isavailable from AbD Serotec.

In an embodiment, A-L- is a species of the formulathio-(CH₂)_(n)-(—O—CH₂—CH₂—)_(m)-D-, wherein n is from 1 to 30 and m isfrom 0 to 10 and D is a group that binds to B; optionally n, m and D maybe as defined above, and thio is selected from —S— and HS—.

In an embodiment, A-L- is a species of the formulathio-(CH₂)_(n)-(—O—CH₂—CH₂—)_(m)-D-, wherein n is from 1 to 30 and m isfrom 0 to 10 and D is a group that binds to B; optionally n, m and D maybe as defined above, and thio is selected from —S— and HS—.

In an embodiment, A-L- is a species of the formulathio-(CH₂)_(n)-(—O—CH₂—CH₂—)_(m)-D-, wherein n is from 1 to 30 and m isfrom 0 to 10 and D is a group that binds to B; optionally n, m and D maybe as defined above, and thio is selected from —S— and HS—.

B is preferably capable of binding selectively to the target species,e.g. C-reactive protein. B preferably comprises or is a binding speciesselected from an antibody, an antibody fragment, an aptamer, anoligosaccharide, a peptide, and a protein. B preferably comprises or isa binding species selected from one or more of an antibody, an antibodyfragment, a nucleic acid and a peptide. Preferably, the probe moietiesbind selectively to C-reactive protein.

If B comprises or is an antibody or an antibody fragment, the antibodyor the antibody fragment may be selected from one or more of the classesIgA, IgD, IgE, IgG and IgM. The antibody or antibody fragment preferablybinds selectively to C-reactive protein.

If B comprises or is an aptamer, the aptamer may be selected from apeptide aptamer, a DNA aptamer and a RNA aptamer.

In an embodiment, an electrode as described herein, e.g. having probemolecules thereon, may be produced by providing the electrode having theplanar surface, then forming a self-assembling monolayer of linkerspecies on the planar surface, and attaching probe moieties, e.g.antibodies, that bind to the target species to at least some of thelinker species. The linker species may optionally be activated, e.g. byreaction with an activator, such as N-hydroxysuccinimde (NHS), to allowfor facile attachment of the probe moieties to the linker species. In anembodiment, the linker species forming the self-assembling monolayer areof the formula A--L, wherein A is a moiety that binds to the surface ofthe electrode, L is a linker moiety capable of binding to a moiety(which may be denoted B) which binds to the target species, e.g. anantibody. In an embodiment, a may be as defined above, and the linkerspecies L forming the monolayer, prior to binding to the probe moieties,is of the formula —(CH₂)_(n)-(—O—CH₂—CH₂—)_(m)-D-, wherein n is from 1to 30 and m is from 0 to 10 and D is a group that binds to B. D may beselected from a single bond, —(C═O)—H, —(C═O)OH—OCH₂—(C═O)H,—OCH₂—(C═O)OH, —(C═O)—NHH, —OCH₂—(C═O)—NH₂, —OCH₂—(C═O)—OH, —OH, —NH₂.

The present application also relates to a method for detecting a targetspecies (including, but not limited to, C-reactive protein) in anelectrochemical impedance spectroscopy technique, wherein the methodcomprises contacting an electrode defined in the first aspect with acarrier medium comprising the target species (including, but not limitedto, C-reactive protein), and detecting an electrical signal at theworking electrode.

Electrochemical impedance spectroscopy (EIS) is known to the skilledperson. Generally, a varying ac potential is applied on a bias (or DC)potential between a working electrode and a counter electrode.Generally, EIS involves scanning across a range of ac frequencies. Theratio of the input signal (typically the varying potential) to theoutput signal (typically the varying current) allows the impedance to becalculated. There is generally a phase difference between the inputsignal and the output signal, such that the impedance can be consideredas a complex function, having a real part (sometimes termed Z′) and animaginary part (sometimes termed Z″). The real and imaginary parts ofimpedance can be plotted against one another, e.g. in the form of aNyquist plot, as illustrated in FIG. 1. By fitting the impedance data toan assumed equivalent circuit, the electron transfer resistance can bedetermined. In the present application, as mentioned above, an idealRandles equivalent circuit can be used to determine the electrontransfer resistance. The frequency range of the varying ac potentialapplied may be from 0.05 Hz to 10 kHz. The amplitude of the applied acpotential, which is typically in the form of a sine wave, may be from 1mV to 100 mV, optionally from 5 mV to 50 mV, optionally from 5 mV to 20mV, optionally from 5 mV to 15 mV, optionally 8 mV to 12 mV, optionallyabout 10 mV. The bias potential (or direct current potential) may be setat any desired potential. If a redox probe is present in the carriermedium, the bias potential may be set at the electrode potential of theredox probe under the conditions at which the method is carrier out.

A redox probe may be present in the carrier medium. The redox probe maybe a transition metal species, wherein the transition metal can adopttwo valence states (e.g. a metal ion (M) being able to adopt M(II) andM(III) states). In an embodiment, the redox probe contains a metal ion,wherein the metal of the metal ion is selected from iron, ruthenium,iridium, osmium, cobalt, tungsten and molybdenum. In an embodiment, theredox probe is selected from Fe(CN)₆ ^(3−/4−), Fe(NH₃)₆ ^(3+/2+),Fe(phen)₃ ^(3+/2+), Fe(bipy)₂ ^(3+/2+), Fe(bipy)₃ ^(3+/2+), Ru^(3+/2+),RuO₄ ^(3−/2−), Ru(CN)₆ ^(3−/4−), Ru(NH₃)₆ ^(3+/2+), Ru(en)₃ ^(3+/2+),Ru(NH₃)₅(Py)^(3+/2+), Ir^(4+/3+), Ir(Cl)₆ ^(2−/3−), Ir(Br)₆ ^(2−/3−),Os(bipy)₂ ^(3+/2+), Os(bipy)₃ ^(3+/2+), OxCl₆ ^(2−/3−), Co(NH₃)₆^(3+/2+), W(CN)₆ ^(3−/4−), Mo(CN)₆ ^(3−/4−), optionally substitutedferrocene, polyferrocene, quiniones, such as p-benzoquinone andhydroquinone and phenol In an embodiment, the redox probe is aniron-containing species in which iron is in Fe(II) and/or Fe(III)states. In an embodiment, the redox probe is Fe(CN)₆ ^(3−/4−). The redoxprobe may be present in the carrier medium an amount of from 0.1 mM to100 mM, optionally from 0.5 mM to 10 mM, optionally from 0.5 mM to 2 mM,optionally from 0.5 mM to 1.5 mM, optionally about 1 mM.

The carrier medium is preferably in liquid form. The carrier liquid maybe any liquid in which the target species (including, but not limitedto, C-reactive protein) can be suspended or dissolved. In an embodiment,the carrier liquid comprises water. In an embodiment, the carrier liquidcomprises a biological fluid. A biological fluid may be a fluid that hasbeen obtained from a subject, which may be a human or an animal. In anembodiment, the carrier liquid comprises an undiluted biological fluid.An undiluted biological fluid in the present context is a biologicalfluid obtained from a subject, e.g. a human or animal, that has not beendiluted with another liquid, although additives such as a redox probe,may be present in the undiluted biological fluid. The biological fluidmay be selected from blood, urine, tears, saliva, sweat, andcerebrospinal fluid.

Optionally, the carrier medium comprises a biological fluid obtainedfrom a subject, e.g. a human or animal, and a diluent. The diluent maybe added to the biological fluid after it has been obtained from thesubject. The diluent may include a liquid medium, e.g. a liquid mediumselected from water and an alcohol, e.g. an alkanol, e.g. ethanol. Thecarrier medium may further comprise a buffer. The buffer may comprise aphosphate.

The method may comprise calculating the concentration of the targetspecies (e.g. C-reactive protein) from the electrical signal. Theelectrical signal may be converted into impedance data and thenconverted to the concentration of the target species (e.g. C-reactiveprotein) from the electrical signal. The electrical signal may beconverted into charge transfer resistance data and then converted to theconcentration of the target species (e.g. C-reactive protein) from theelectrical signal. The method may involve comparing the data obtained inthe electrochemical impedance spectroscopy technique, e.g. from theelectrical signal, the impedance data or the charge transfer resistancedata, and comparing the data with data obtained in a calibration step,to obtain the concentration of the target species (e.g. C-reactiveprotein). The method may involve an initial calibration step thatdetermines a relationship between the concentration of the targetspecies (e.g. C-reactive protein) and electrochemical data obtained fromthe electrochemical signal in the electrochemical impedance spectroscopytechnique; the electrochemical data may be selected from impedance dataand charge transfer resistance data; the relationship may be used toconvert the electrochemical data obtained from a sample of interest inthe electrochemical impedance spectroscopy technique to theconcentration of the target species (e.g. C-reactive protein) in thesample.

The concentration of the target species (e.g. C-reactive protein) in thecarrier medium may be 0.1 nM or more, optionally 0.16 nM or more,optionally 0.2 nM or more, optionally 0.5 nM or more. The concentrationof the target species (e.g. C-reactive protein) in the carrier mediummay be 100 nM or less, optionally 80 nM or less, optionally 50 nM orless, optionally 10 nM or less. The concentration of the target species(e.g. C-reactive protein) in the carrier medium may be from 0.1 nM to100 nM, optionally from 0.16 nM to 100 nM, optionally from 0.16 nM to 50nM.

The concentration of the target species (e.g. C-reactive protein) in thecarrier medium may be 10 μg/L or more, optionally 15 μg/L or more,optionally 19 μg/L or more, optionally 20 μg/L or more, optionally 0.1mg/L or more, optionally 1 mg/L or more, optionally 3 mg or more. Theconcentration of the target species (e.g. C-reactive protein) in thecarrier medium may be 100 mg/L or less, optionally 80 mg/L or less,optionally 50 mg/L or less, optionally 30 mg/L or less. Theconcentration of the target species (e.g. C-reactive protein) in thecarrier medium may be from 10 μg/L to 100 mg/L, optionally from 19 μg/Lto 100 mg/L, optionally from 19 μg/L to 50 mg/L.

The calculating may comprise one or more comparisons of the electricalsignal with the electrical signal of an ideal equivalent circuit.

The present inventors have found that they can regenerate the electrode,that has been bound to target species (e.g. C-reactive protein), bydissociating bound target species (e.g. C-reactive protein) from theelectrode. The method may involve, after the contacting of the electrodewith the target species (e.g. C-reactive protein), such that the targetspecies (e.g. C-reactive protein) is bound to the probe molecules, anddetecting the electrical signal, dissociating the target species (e.g.C-reactive protein) from the probe molecules. The dissociating maycomprise contacting of the electrode surface having target species (e.g.C-reactive protein) thereon with an alkali liquid medium, e.g. an alkaliaqueous liquid medium, optionally having a pH of 8 or more, optionally apH of 9 or more, optionally a pH of 10 or more, optionally a pH of 11 ormore, optionally a pH of 8 to 12, optionally a pH of 9 to 12. The alkaliliquid medium may contain a basic substance. The basic substance ispreferably soluble in water. The basic substance may be selected from,but not limited to, a metal hydroxide, a metal carbonate, ammonia. Themetal of the metal hydroxide or metal carbonate may be selected fromGroup 1 and Group 2 of the Periodic Table.

The present invention also relates to an electrochemical impedancespectrometer, wherein the spectrometer comprises an electrode as definedherein. The electrochemical impedance spectrometer may be of a standarddesign. The electrochemical impedance spectrometer may comprise anelectrode of the first aspect as a working electrode, a counterelectrode, and, if desired a reference electrode. The electrochemicalimpedance spectrometer preferably comprises a means for applying,controlling and varying a potential between the working and counterelectrodes, and a means for measuring the resultant current. Theelectrochemical impedance spectrometer preferably comprises apotentiostat for controlling the potential and measuring the resultantcurrent. The electrochemical impedance spectrometer preferably comprisesa means for calculating impedance data from the potential applied andthe resultant current. The electrochemical impedance spectrometer maycomprise a means for calculating electron transfer resistance of theworking electrode.

The present invention also relates to the use of an electrode asdescribed herein or an electrochemical impedance spectrometer asdescribed herein for the detection of a target species, e.g. C-reactiveprotein. The use may include detecting the presence of and/or detectingthe concentration of the target species, e.g. C-reactive protein.

Examples

In the examples below, human CRP, human blood serum and bovine serumalbumin (BSA) were purchased from Sigma Aldrich. The goat anti-human CRPpolyclonal antibody was purchased from AbD Serotec.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich.Polyethylene glycol (PEG) thiol HS—C₁₁-(EG)₃-OCH₂—COOH was purchasedfrom Prochimia Surfaces, Poland. Ultrapure water (18.2 MΩ/cm) wasobtained from a Milli-Q system and used throughout. Phosphate bufferedsaline (PBS) with Tween-20 (PBST, 10 mM, pH 7.4) was prepared bydissolving PBS tablets (Sigma Aldrich) in water with 0.2% v/v Tween-20added, and filtered using a 0.22 μm membrane filter. All other chemicalswere of analytical grade.

2.2. Apparatus

Electrochemical experiments were performed on an Autolab Potentiostat 12equipped with an FRA2 module (Metrohm Autolab B.V.). A conventionalthree-electrode system with a gold disk working electrode (1.6 mmdiameter, BASi), platinum wire counter electrode and a silver/silverchloride (Ag/AgCl) reference electrode (CH Instruments) were used. Allpotentials are reported relative to this reference. CRP stock solutionconcentrations were calculated via the UV absorbance at 280 nm (Motie etal. 1996) using a Shimadzu UV spectrometer (Shimadzu ScientificInstruments).

2.3. Surface Preparation

Gold electrodes were firstly polished with 3.0, 1.0 and 0.1 μm diamondspray (Kemet International Ltd) in sequence and ultrasonically washed inwater for about 5 min prior to immersion in freshly prepared piranhasolution (concentrated H₂SO₄: H₂O₂, v/v 3:1. Caution: this must behandled with extreme care!) for 15 min. Electrodes were thenelectrochemically polished by potential cycling (CV) between −0.1 and1.25 V until a stable reduction peak was obtained. The effective surfacearea of the gold electrode can be calculated at this point by taking thearea of the cathodic peak in coulombs and applying the relationship

$A = \frac{Q}{482\mspace{14mu} {\mu C}\mspace{14mu} {cm}^{- 2}}$

(Hoogvliet et al. 2000), and the reported charge transfer resistance(R_(ct)) was normalized by this.

(1) Hoogvliet, J. C.; Dijksma, M.; Kamp, B.; van Bennekom, W. P. AnalChem 2000, 72, 2016.

Pre-treated gold electrodes were then dried in a flow of nitrogen gasand immediately immersed in a 10 mM solution ofHS—C₁₁H₂₂-(EG)₃-OCH₂—COOH in ethanol for 16 hours at room temperature.The biocompatible (Cho et al. 2011, Klapshina et al. 2010) andantifouling properties (Harder et al. 1998, Schilp et al. 2009) of suchfilms are sufficient to enable specific assessments to be made incomplex biological fluid. After SAM formation gold surfaces were rinsedwith ethanol then water and dried in a flow of nitrogen gas prior toincubation in a solution containing 0.4 M EDC and 0.1 M NHS for 15minutes (terminal carboxyl group activation) and then 10 μM CRP antibodysolution (PBST, pH 7.4) for 1 hour (FIG. 7), unless stated otherwise.

2.4. Electrochemical Impedance Spectroscopy

EIS spectra were recorded across a 0.05 Hz to 10 kHz frequency range.The amplitude of the applied sine wave potential was 10 mV with thedirect current potential set at 0.25 V (the E₀ of the redox probe used,1.0 mM Fe(CN)₆ ^(3−/4−)). Data was acquired in 10 mM PBST solution,plotted in the form of complex plane diagrams (Nyquist plots), andfitted through an ideal Randles equivalent circuit (Vyas et al. 2010),as illustrated in FIG. 8. In this figure, R_(ct) represents the charge(or electron) transfer resistance, Z_(w) represents the Warburgimpedance, R_(s) is the solution resistance, and Cal is the capacitancebetween the electrode surface and target species or ions in the liquidcarrier medium. The raw impedance data is acquired by a FRA (frequencyresolved analyser) module (in this case manufactured by Autolab, TheNetherlands), then fit to the equivalent circuit in FIG. 8 using inbuiltsoftware (“Fit and Simulation version 1.7”). The software runs multipleiterations of fittings to reduce errors then outputs values for allequivalent circuit components, including Rct.

Assays were carried out by electrode incubation in CRP spiked PBST,specific dilutions of blood serum or whole blood serum at roomtemperature for 30 min each. In the first two cases PBST was pre-dopedwith redox probe. EIS responses were normally recorded in the sameincubation solution, with the exception of the whole (undiluted) bloodserum tests, where the electrodes were rinsed with PBST after incubationprior to assessment in PBST containing 1.0 mM Fe(CN)₆ ^(3−/4−). Toinitially evaluate interfacial selectivity BSA was used. Used surfaceswere regenerated using 6 mM NaOH and 0.6% ethanol for 5 min (Albrecht etal. 2008) prior to PBST washing.

3. Results and Discussion 3.1. Biosensor Fabrication and InitialImpedance

EIS presents a useful means of characterising the stepwise fabricationof a receptive surface. In Nyquist plots, the semicircles at lowsampling frequency report on charge transfer restrictions imposedsterically or electrostatically as films are constructed. Predictably,there are sharp increases in R_(CT) as the receptor layer is fabricated.R_(ct) specifically increases from less than 4.5 kΩ/cm² to ˜45 MΩ/cm²after the formation of the PEG SAM and further upwards on antibodyimmobilisation (FIG. 1). The resistance of the interface thereafterresponds in a calibratable manner to target protein binding.

The present inventors found that the initial resistance of an electrode,i.e. before binding to CRP, plays a role in subsequently observedsensitivity. This initial resistance is directly tuneable through theantibody surface density. This is itself controllable through eitherincubation time or incubation concentration as the layer is constructed.FIGS. 2A-2B summarise the observed trend in assay sensitivity withinitial layer charge transfer resistance. Notably, as the antibodysurface coverage decreases (as the immobilization timeframe is reducedfrom 70 min to 10 min), assay sensitivity initially increases inmagnitude by up to 400% (and the limit of detection decreasesconsequently) before falling presumably as the density of surface boundand functional antibodies falls below the point where specific targetbinding is effective (in terms of probability). These observations arerobust and reproducible across numerous assays.

3.2. Detection of CRP in Buffer

The prepared interfaces were subsequently used to screen CRP in PBST.From the progressive and then saturating increases in R_(CT) withconcentration (FIGS. 3A-3B), a dissociation constant K_(D) of 1.1±0.11nM, can be derived, a value in excellent agreement with a previousdifferential pulse voltammetry determination with the same polyclonalantibody (Hennessey et al. 2009).

Prior to saturation, R_(CT) reports linearly with logarithmicsensitivity on CRP concentration across a 0.5-50 nM range (equivalent to60 μg/L to 6.0 mg/L) with a limit of detection (LOD) of 176±18 pM. Thislow detection limit (equating to ˜19 μg/L) confirms sensitivity to becomfortably sufficient for practical application and is married to aassay range encompassing that which is clinically relevant. This range,taken with its associated detection limit, exceeds the clinicalrelevance of any prior reported CRP assay to the best of our knowledge.The prepared interfaces are, additionally, unresponsive (<3% change insignal) to BSA levels of up to 100 nM.

3.3. Detection of CRP in the Blood Serum

From a point of care perspective, the direct and facile assessment ofCRP in blood serum is necessary. In any label free assay, however, thisis exceedingly demanding. Though a number of amperometric or sandwichbased EIS immunoassay methods have been demonstrated (Balkenhohl andLisdat 2007a, b, Pan et al. 2010, Rosales-Rivera et al. 2011, Tran etal. 2011), to the best of our knowledge, there exists no prior report ofa non-amplified and label free impedance assays that has been reportedas being effective in undiluted complex biological media.

Being confident about the degree of control we had over our electrodeinterfaces and in the light of the low levels of response to even highlevels of BSA, we screened here for CRP in blood serum in two ways. Inthe first instance, in situ assessments were made with CRP spiked bloodserum at controllable dilution in PBST (FIGS. 4A-4B). Under suchcircumstances reliable linear assessments were possible across theclinically relevant range with an LOD of 262±28 pM at serumconcentrations of up to 10% v/v.

Subsequent analyses were carried out with CRP spiked undiluted bloodserum; Resistance optimized sensor electrodes were incubated in thesesolutions for 15 minutes and then measured after rinsing with PBST. Asis evident in FIG. 5, the sensor response in such analyses is markedlyclose (in the higher concentration range of most clinical relevance,10-50 nM, the differences are <3%) to that observed in spiked PBST. Thisenables analysis to be performed in whole blood serum across the CRPconcentration range required for useful cardiovascular disease riskassessment. From a practical perspective, these assays can be carriedout with as little as 5 μL of undiluted blood serum and reportquantitatively within 10 min. The utilised electrode interfaces arealso, subsequently, reusable (see below).

3.4. Biosensor Regeneration

Surface regeneration was achieved with high fidelity (see FIG. 6) byimmersion of used surfaces in 6 mM NaOH and 0.6% ethanol for 5 min (todisassociate the CRP antibody-antigen complex) and then washing withPBST. We believe the robust regeneration is possible partially becauseof the absence of the BSA passivation commonly employed at suchimmunoassaying surfaces. The interfaces herein could be reused withoutsignificant detriment of the assay (97% of the original interfaceresponse retained over 7 regenerations). This regeneration is effectivefor assays carried out in PBS, diluted serum or whole serum.

The present inventors have found an optimised and reusableelectrochemical label free biosensor capable of the reliable detectionof CRP across the clinically relevant range in dilute or whole bloodserum. The in situ determined polyclonal antibody binding affinity mapsvery well onto previous determinations at comparable interfaces. Inaddition to facilitating high assay sensitivity and selectivity, theprepared biosensor interfaces also exhibited satisfying reusability. Inoptimising the initial interfacial resistance through antibody surfacedensity, assay sensitivity can be markedly increased.

Assays such as these are easily integrated into portable and multiplexedformats capable of sampling just a few μL of biological fluid withinminutes. We believe the presented results serve as an important basisfor the development of convenient point of care analysis of a marker(i.e. CRP) long considered as a sensitive probe of infection, trauma,inflammation and cardiac risk.

REFERENCES

-   Albrecht C, Kaeppel N, Gauglitz G. 2008. Two immunoassay formats for    fully automated CRP detection in human serum. Analytical and    Bioanalytical Chemistry 391: 1845-1852.-   Balkenhohl T, Lisdat F. 2007a. Screen-printed electrodes as    impedimetric immunosensors for the detection of    anti-transglutaminase antibodies in human sera. Analytica Chimica    Acta 597: 50-57.-   Balkenhohl T, Lisdat F. 2007b. An impedimetric immunosensor for the    detection of autoantibodies directed against gliadins. Analyst 132:    314-322.-   Bogomolova A, Komarova E, Reber K, Gerasimov T, Yavuz O, Bhatt S,    Aldissi M. 2009. Challenges of Electrochemical Impedance    Spectroscopy in Protein Biosensing. Analytical Chemistry 81:    3944-3949.-   Buch M, Rishpon J. 2008. An Electrochemical Immunosensor for    C-Reactive Protein Based on Multi-Walled Carbon Nanotube-Modified    Electrodes. Electroanalysis 20: 2592-2594.-   Centi S, Sanmartin L B, Tombelli S, Palchetti I, Mascini M. 2009.    Detection of C Reactive Protein (CRP) in Serum by an Electrochemical    Aptamer-Based Sandwich Assay. Electroanalysis 21: 1309-1315.-   Chen X J, Wang Y Y, Zhou J J, Yan W, Li X H, Zhu J J. 2008.    Electrochemical impedance immunosensor based on three-dimensionally    ordered macroporous gold film. Analytical Chemistry 80: 2133-2140.-   Cho H Y, et al. 2011. Synthesis of Biocompatible PEG-Based Star    Polymers with Cationic and Degradable Core for siRNA Delivery.    Biomacromolecules 12: 3478-3486.-   Gabay C, Kushner I. 1999. Mechanisms of disease: Acute-phase    proteins and other systemic responses to inflammation. New England    Journal of Medicine 340: 448-454.-   Harder P, Grunze M, Dahint R, Whitesides G M, Laibinis P E. 1998.    Molecular conformation in oligo(ethylene glycol)-terminated    self-assembled monolayers on gold and silver surfaces determines    their ability to resist protein adsorption. Journal of Physical    Chemistry B 102: 426-436.-   Hennessey H, Afara N, Omanovic S, Padjen A L. 2009. Electrochemical    investigations of the interaction of C-reactive protein (CRP) with a    CRP antibody chemically immobilized on a gold surface. Analytica    Chimica Acta 643: 45-53.-   Hoogvliet J C, Dijksma M, Kamp B, van Bennekom W P. 2000.    Electrochemical pretreatment of polycrystalline gold electrodes to    produce a reproducible surface roughness for self assembly: A study    in phosphate buffer pH 7.4. Analytical Chemistry 72: 2016-2021.-   Hu W P, Hsu H Y, Chiou A, Tseng K Y, Lin H Y, Chang G L, Chen    S J. 2006. Immunodetection of pentamer and modified C-reactive    protein using surface plasmon resonance biosensing. Biosensors &    Bioelectronics 21: 1631-1637.-   Kim N, Kim D K, Cho Y J. 2009. Development of indirect-competitive    quartz crystal microbalance immunosensor for C-reactive protein.    Sensors and Actuators B-Chemical 143: 444-448.-   Klapshina L G, Douglas W E, Grigoryev I S, Ladilina E Y, Shirmanova    M V, Mysyagin S A, Balalaeva I V, Zagaynova E V. 2010. Novel    PEG-organized biocompatible fluorescent nanoparticles doped with an    ytterbium cyanoporphyrazine complex for biophotonic applications.    Chemical Communications 46: 8398-8400.-   Kushner I, Sehgal A R. 2002. Is high-sensitivity C-reactive protein    an effective screening test for cardiovascular risk? Archives of    Internal Medicine 162: 867-869.-   Lee W B, Chen Y H, Lin H I, Shiesh S C, Lee G B. 2011. An integrated    microfluidic system for fast, automatic detection of C-reactive    protein. Sensors and Actuators B-Chemical 157: 710-721.-   May A, Wang T J. 2007. Evaluating the role of biomarkers for    cardiovascular risk prediction: focus on CRP, BNP and urinary    microalbumin. Expert Review of Molecular Diagnostics 7: 793-804.-   Meyer M H F, Hartmann M, Keusgen M. 2006. SPR-based immunosensor for    the CRP detection—A new method to detect a well known protein.    Biosensors & Bioelectronics 21: 1987-1990.-   Miller V M, Redfield M M, McConnell J P. 2007. Use of BNP and CRP as    biomarkers in assessing cardiovascular disease: Diagnosis versus    risk. Current Vascular Pharmacology 5: 15-25.-   Motie M, Brockmeier S, Potempa L A. 1996. Binding of model soluble    immune complexes to modified C-reactive protein. Journal of    Immunology 156: 4435-4441.-   Mygind N D, Harutyunyan M J, Mathiasen A B, Ripa R S, Thune J J,    Gotze J P, Johansen J S, Kastrup J, Grp C T. 2011. The influence of    statin treatment on the inflammatory biomarkers YKL-40 and HsCRP in    patients with stable coronary artery disease. Inflammation Research    60: 281-287.-   Pai J K, Mukamal K J, Rexrode K M, Rimm E B. 2008. C-Reactive    Protein (CRP) Gene Polymorphisms, CRP Levels, and Risk of Incident    Coronary Heart Disease in Two Nested Case-Control Studies. Plos One    3.-   Pan Y, Sonn G A, Sin M L Y, Mach K E, Shih M C, Gau V, Wong P K,    Liao J C. 2010. Electrochemical immunosensor detection of urinary    lactoferrin in clinical samples for urinary tract infection    diagnosis. Biosensors & Bioelectronics 26: 649-654.-   Pearson T A, Mensah G A, Hong Y L, Smith S C. 2004. CDC/AHA Workshop    on Markers of Inflammation and Cardiovascular Disease—Application to    Clinical and Public Health Practice—Overview. Circulation 110:    E543-E544.-   Qureshi A, Gurbuz Y, Kallempudi S, Niazi J H. 2010. Label-free RNA    aptamer-based capacitive biosensor for the detection of C-reactive    protein. Physical Chemistry Chemical Physics 12: 9176-9182.-   Roberts W L, Sedrick R, Moulton L, Spencer A, Rifai N. 2000.    Evaluation of four automated high-sensitivity C-reactive protein    methods: Implications for clinical and epidemiological applications.    Clinical Chemistry 46: 461-468.-   Roberts W L, Moulton L, Law T C, Farrow G, Cooper-Anderson M, Savory    J, Rifai N. 2001. Evaluation of nine automated high-sensitivity    c-reactive protein methods: Implications for clinical and    epidemiological applications. Part 2. Clinical Chemistry 47:    418-425.-   Rodriguez M C, Kawde A N, Wang J. 2005. Aptamer biosensor for    label-free impedance spectroscopy detection of proteins based on    recognition-induced switching of the surface charge. Chemical    Communications: 4267-4269.-   Rosales-Rivera L C, Acero-Sanchez J L, Lozano-Sanchez P, Katakis I,    O'Sullivan C K. 2011. Electrochemical immunosensor detection of    antigliadin antibodies from real human serum. Biosensors &    Bioelectronics 26: 4471-4476.-   Schilp S, Rosenhahn A, Pettitt M E, Bowen J, Callow M E, Callow J A,    Grunze M. 2009. Physicochemical Properties of (Ethylene    Glycol)-Containing Self-Assembled Monolayers Relevant for Protein    and Algal Cell Resistance. Langmuir 25: 10077-10082.-   Tran D T, Vermeeren V, Grieten L, Wenmackers S, Wagner P, Pollet J,    Janssen K P P, Michiels L, Lammertyn J. 2011. Nanocrystalline    diamond impedimetric aptasensor for the label-free detection of    human IgE. Biosensors & Bioelectronics 26: 2987-2993. Vermeeren V,    Grieten L, Vanden Bon N, Bijnens N, Wenmackers S, Janssens S D,    Haenen K, Wagner P, Michiels L. 2011. Impedimetric, diamond-based    immunosensor for the detection of C-reactive protein. Sensors and    Actuators B-Chemical 157: 130-138.-   Vikholm-Lundin I, Albers W M. 2006. Site-directed immobilisation of    antibody fragments for detection of C-reactive protein. Biosensors &    Bioelectronics 21: 1141-1148.-   Vyas R N, Li K Y, Wang B. 2010. Modifying Randles Circuit for    Analysis of Polyoxometalate Layer-by-Layer Films. Journal of    Physical Chemistry B 114: 15818-15824.-   Wee K W, Kang G Y, Park J, Kang J Y, Yoon D S, Park J H, Kim    T S. 2005. Novel electrical detection of label-free disease marker    proteins using piezoresistive self-sensing micro-cantilevers.    Biosensors & Bioelectronics 20: 1932-1938.

1. An electrode for use in the electrochemical detection of a targetspecies, the electrode comprising: a planar surface having probemolecules disposed thereon, in which the probe molecules are capable ofelectrochemically detecting a target species by binding selectively tothe target species, wherein the electrode, prior to binding of the probemolecules with the target species, has an electron transfer resistanceper area of the electrode of from 10 megaohms cm⁻² to 95 megaohms cm⁻²,and wherein the target species is an antigen or analyte that isindicative of a particular disease.
 2. An electrode according to claim1, wherein the target species is an antigen or analyte selected from thegroup consisting of angiotensin I converting enzyme(peptidyl-dipeptidase A) 1; adiponectin; advanced glycosylation endproduct-specific receptor; alpha-2-HS-glycoprotein; angiogenin,ribonuclease, RNase A family, 5; apolipoprotein A-1; apolipoprotein B(including Ag(x) antigen); apolipoprotein E; BCL2-associated X protein;B-cell CLL/lymphoma 2; complement C3; chemokine (C-C motif) ligand 2; CD14, soluble; CD 40, soluble; cdk5; pentraxin-related; cathepsin B;dipeptidyl peptidase IV; Epidermal growth factor; endoglin; Fas;fibrinogen; ferritin; growth hormone 1; alanine aminotransferase;hepatocyte growth factor; haptoglobin; heat shock 70 kDa protein 1 B;intercellular adhesion molecule 1; insulin-like growth factor 1(somatomedin C); insulin-like growth factor 1 receptor; insulin-likegrowth factor binding protein 1; insulin-like growth factor bindingprotein 2; insulin-like growth factor-binding protein 3; interleukin 18;interleukin 2 receptor, alpha; interleukin 2 receptor, beta; interleukin6 (interferon, beta 2); interleukin 6 receptor; interleukin 6 signaltransducer (gp130, oncostatin M receptor); interleukin 8; activin A;leptin (obesity homolog, mouse); plasminogen activator, tissue;proopiomelanocortin(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulatinghormone/beta-melanocyte stimulating hormone/beta-endorphin); proinsulin;resistin; selectin e (endothelial adhesion molecule 1); selectin P(granule membrane protein 140 kDa, antigen CD62); serpin peptidaseinhibitor, clade E (nexin, plasminogen activator inhibitor type 1),member 1; serum/glucocorticoid regulated kinase; sex hormone-bindingglobulin; transforming growth factor, beta 1 (Camurati-Engelmanndisease); TIMP metallopeptidase inhibitor 2; tumor necrosis factorreceptor superfamily, member 1 B; vascular cell adhesion molecule 1(VCAM-1); vascular endothelial growth factor; Factor II, Factor V,Factor VIII, Factor IX, Factor XI, Factor XII, F/fibrin degradationproducts, thrombin-antithrombin III complex, fibrinogen, plasminogen,prothrombin, and von Willebrand factor and the like. Markers useful fordiabetes include for example C-reactive protein; glucose; insulin; TRIG;GPT; HSPA1 B; IGFBP2; LEP; ADIPOQ; CCL2; ENG; HP; IL2RA; SCp; SHBG; andTIMP2.
 3. An electrode according to claim 1, wherein the probe moleculescomprise at least one of: antibodies or antibody fragments.
 4. Anelectrode according to claim 1, wherein the electrode prior to bindingof the probe molecules with the target species has an electron transferresistance per area of the electrode of from 55 megaohms cm⁻² to 95megaohms cm⁻².
 5. An electrode for use according to claim 1, wherein theelectrode comprises a metal selected from the group consisting of: gold,rhenium, iridium, palladium, platinum, copper, indium, rubidium silver,and combinations thereof.
 6. An electrode according to claim 1, whereinthe probe molecule is of the formula A-L-B, where A is a moiety thatbinds to the surface of the electrode, L is a linker moiety and B is amoiety which binds selectively to the target species.
 7. An electrodeaccording to claim 6, wherein A is selected from the group consistingof: biotin, hydrazine, alkynyl, alkylazide, amino, hydroxyl, carboxy,thiol, aldehyde, phosphoinothioester, maleimidyl, succinyl,succinimidyl, isocyanate, ester, strepavidin, avidin, neuavidin, biotinbinding proteins, and combinations thereof.
 8. An electrode for use inthe electrochemical detection of a target species, the electrodecomprising: a planar surface having probe molecules disposed thereon, inwhich the probe molecules are capable of electrochemically detecting atarget species by binding selectively to the target species, wherein theelectrode, prior to binding of the probe molecules with the targetspecies, has an electron transfer resistance per area of the electrodeof from 10 megaohms cm⁻² to 95 megaohms cm⁻², wherein the target speciesis an antigen or analyte that is indicative of a particular disease, andwherein the probe molecule is of the formula A-L-B, where A is a moietythat binds to the surface of the electrode, L is a linker moiety and Bis a moiety which binds selectively to the target species, and Lcomprises an alkylene moiety comprising at least 10 carbons.
 9. Anelectrode according to claim 8, wherein the target species is an antigenor analyte selected from the group consisting of angiotensin Iconverting enzyme (peptidyl-dipeptidase A) 1; adiponectin; advancedglycosylation end product-specific receptor; alpha-2-HS-glycoprotein;angiogenin, ribonuclease, RNase A family, 5; apolipoprotein A-1;apolipoprotein B (including Ag(x) antigen); apolipoprotein E;BCL2-associated X protein; B-cell CLL/lymphoma 2; complement C3;chemokine (C-C motif) ligand 2; CD 14, soluble; CD 40, soluble; cdk5;pentraxin-related; cathepsin B; dipeptidyl peptidase IV; Epidermalgrowth factor; endoglin; Fas; fibrinogen; ferritin; growth hormone 1;alanine aminotransferase; hepatocyte growth factor; haptoglobin; heatshock 70 kDa protein 1 B; intercellular adhesion molecule 1;insulin-like growth factor 1 (somatomedin C); insulin-like growth factor1 receptor; insulin-like growth factor binding protein 1; insulin-likegrowth factor binding protein 2; insulin-like growth factor-bindingprotein 3; interleukin 18; interleukin 2 receptor, alpha; interleukin 2receptor, beta; interleukin 6 (interferon, beta 2); interleukin 6receptor; interleukin 6 signal transducer (gp130, oncostatin Mreceptor); interleukin 8; activin A; leptin (obesity homolog, mouse);plasminogen activator, tissue; proopiomelanocortin(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulatinghormone/beta-melanocyte stimulating hormone/beta-endorphin); proinsulin;resistin; selectin e (endothelial adhesion molecule 1); selectin P(granule membrane protein 140 kDa, antigen CD62); serpin peptidaseinhibitor, clade E (nexin, plasminogen activator inhibitor type 1),member 1; serum/glucocorticoid regulated kinase; sex hormone-bindingglobulin; transforming growth factor, beta 1 (Camurati-Engelmanndisease); TIMP metallopeptidase inhibitor 2; tumor necrosis factorreceptor superfamily, member 1 B; vascular cell adhesion molecule 1(VCAM-1); vascular endothelial growth factor; Factor II, Factor V,Factor VIII, Factor IX, Factor XI, Factor XII, F/fibrin degradationproducts, thrombin-antithrombin III complex, fibrinogen, plasminogen,prothrombin, and von Willebrand factor and the like. Markers useful fordiabetes include for example C-reactive protein; glucose; insulin; TRIG;GPT; HSPA1 B; IGFBP2; LEP; ADIPOQ; CCL2; ENG; HP; IL2RA; SCp; SHBG; andTIMP2.
 10. An electrode according to claim 1, wherein the probe moleculeis of the formula A-L-B, where A is a moiety that binds to the surfaceof the electrode, L is a linker moiety and B is a moiety which bindsselectively to the target species, and L comprises an alkylene moietycomprising at least 10 carbons in which. L is of the formula—(CH₂)_(n)-(—O—CH₂—CH₂)_(m)-D-, wherein n is from 1 to 30 and m is from0 to 10 and D is a group that binds to B.
 11. (canceled)
 12. (canceled)13. A method for detecting a target species in an electrochemicalimpedance spectroscopy technique, wherein the method comprises:contacting an electrode with a carrier medium comprising a targetspecies in which the electrode comprises a planar surface having probemolecules disposed thereon which are capable of electrochemicallydetecting the target species via selectively binding to the targetspecies, wherein the electrode, prior to selective binding of the probemolecules with the target species, has an electron transfer resistanceper area of the electrode of from 10 megaohms cm⁻² to 95 megaohms cm⁻²;and detecting an electrical signal at the electrode to electrochemicallydetect the target species, wherein the target species is an antigen oranalyte that is indicative of a particular disease.
 14. A methodaccording to claim 13, wherein the target species is an antigen oranalyte selected from the group consisting of angiotensin I convertingenzyme (peptidyl-dipeptidase A) 1; adiponectin; advanced glycosylationend product-specific receptor; alpha-2-HS-glycoprotein; angiogenin,ribonuclease, RNase A family, 5; apolipoprotein A-1; apolipoprotein B(including Ag(x) antigen); apolipoprotein E; BCL2-associated X protein;B-cell CLL/lymphoma 2; complement C3; chemokine (C-C motif) ligand 2; CD14, soluble; CD 40, soluble; cdk5; pentraxin-related; cathepsin B;dipeptidyl peptidase IV; Epidermal growth factor; endoglin; Fas;fibrinogen; ferritin; growth hormone 1; alanine aminotransferase;hepatocyte growth factor; haptoglobin; heat shock 70 kDa protein 1 B;intercellular adhesion molecule 1; insulin-like growth factor 1(somatomedin C); insulin-like growth factor 1 receptor; insulin-likegrowth factor binding protein 1; insulin-like growth factor bindingprotein 2; insulin-like growth factor-binding protein 3; interleukin 18;interleukin 2 receptor, alpha; interleukin 2 receptor, beta; interleukin6 (interferon, beta 2); interleukin 6 receptor; interleukin 6 signaltransducer (gp130, oncostatin M receptor); interleukin 8; activin A;leptin (obesity homolog, mouse); plasminogen activator, tissue;proopiomelanocortin(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulatinghormone/beta-melanocyte stimulating hormone/beta-endorphin); proinsulin;resistin; selectin e (endothelial adhesion molecule 1); selectin P(granule membrane protein 140 kDa, antigen CD62); serpin peptidaseinhibitor, clade E (nexin, plasminogen activator inhibitor type 1),member 1; serum/glucocorticoid regulated kinase; sex hormone-bindingglobulin; transforming growth factor, beta 1 (Camurati-Engelmanndisease); TIMP metallopeptidase inhibitor 2; tumor necrosis factorreceptor superfamily, member 1 B; vascular cell adhesion molecule 1(VCAM-1); vascular endothelial growth factor; Factor II, Factor V,Factor VIII, Factor IX, Factor XI, Factor XII, F/fibrin degradationproducts, thrombin-antithrombin III complex, fibrinogen, plasminogen,prothrombin, and von Willebrand factor and the like. Markers useful fordiabetes include for example C-reactive protein; glucose; insulin; TRIG;GPT; HSPA1 B; IGFBP2; LEP; ADIPOQ; CCL2; ENG; HP; IL2RA; SCp; SHBG; andTIMP2.
 15. A method according to claim 13, wherein the carrier mediumcomprises a biological fluid.
 16. A method according to claim 15,wherein the biological fluid is selected from the group consisting of:blood, urine, tears, saliva, sweat, cerebrospinal fluid, andcombinations thereof.
 17. A method according to claim 15, wherein thebiological fluid is an undiluted biological fluid.
 18. A methodaccording to claim 13, wherein the carrier medium further comprises aredox probe.
 19. A method according to claim 13, the method additionallycomprising: after contacting the electrode with the carrier mediumcomprising the target species to result in the target species beingbound to the probe molecules, dissociating the target species from theprobe molecules.
 20. A method according to claim 19, the method furthercomprising: after dissociating the target species from the probemolecules, reusing the electrode in an electrochemical method.
 21. Amethod according to claim 13, wherein the method further involves:calculating a concentration of the target species from the electricalsignal.
 22. (canceled)